RESUMEN
Ayahuasca is a brew made of two admixture plants containing dimethyltryptamine (DMT) and b-carbolines (harmine and tetrahydroharmine). The indigenous groups of the Amazonas basin have been using it for centuries as an ethnomedical substance in healing and spiritual-religious rituals. During the last two decades the brew has raised increased scientific and public interest worldwide about its healing effects. Present paper addresses the therapeutic potentials of ayahuasca use and outlines the cellular mechanisms behind - in focus of the Q-1 receptor mediated action of DMT. The scientific investigation of ayahuasca is complicated by methodical problems, legal issues, and sociocultural pre-conceptions.
Asunto(s)
Alucinógenos/uso terapéutico , Harmina/uso terapéutico , N,N-Dimetiltriptamina/uso terapéutico , Extractos Vegetales/uso terapéutico , Alucinógenos/farmacología , Harmina/análogos & derivados , Harmina/farmacología , Humanos , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , N,N-Dimetiltriptamina/farmacología , N,N-Dimetiltriptamina/fisiología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/fisiologíaRESUMEN
OBJECTIVE AND DESIGN: We sought to determine the effect of necrosis-induced activation of the complement protein C3 in medulloblastoma. MATERIALS/METHODS: Twelve medulloblastoma surgical specimens were evaluated for complement activation using immunohistochemistry, with H&E stains performed on adjacent tissue sections to determine the relationship of complement activation to necrotic tissue. Flow cytometry and Western blot were performed on three established medulloblastoma lines and one surgically-procured cell culture to determine expression of C3a receptor (C3aR) in medulloblastoma. In vitro proliferation of siRNA C3aR knockdown cells was compared to that of control siRNA cells with cell line Daoy. RESULTS: Three surgical specimens were found to have necrosis on H&E sections. In each case, iC3b staining was identified on adjacent sections, limited to the necrotic region. In no case did necrosis occur without iC3b staining on adjacent sections. C3aR protein was demonstrated on both the three established cell lines and on the surgical culture. Proliferation assays of Daoy cells with siRNA knockdown vs. control siRNA revealed significantly reduced proliferation at 72 h (p = 0.001). CONCLUSIONS: Necrosis is associated with complement activation in medulloblastoma. Medulloblastoma cells express C3aR, and siRNA-mediated knockdown of C3aR inhibits proliferation of these cells in vitro.
Asunto(s)
Proliferación Celular/fisiología , Neoplasias Cerebelosas/patología , Complemento C3/fisiología , Meduloblastoma/patología , Línea Celular Tumoral , Neoplasias Cerebelosas/fisiopatología , Técnicas de Silenciamiento del Gen , Humanos , Técnicas In Vitro , Meduloblastoma/fisiopatología , Necrosis/patología , ARN Interferente Pequeño/farmacología , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/genética , Receptores de Complemento/fisiología , Transducción de Señal/fisiologíaRESUMEN
Abstract The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.
Asunto(s)
Complemento C1q/efectos de los fármacos , Infecciones/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Receptores de Complemento/efectos de los fármacos , Animales , Humanos , Ligandos , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatología , Receptores de Complemento/química , Receptores de Complemento/genéticaRESUMEN
Monocytes were isolated in high yield (approximately 80%) and purity (greater than 90%) by Percoll gradient centrifugation and incubated in Teflon culture vessels. Using this culture method, we routinely recovered 80% of the cells originally placed into culture. Studies of the C3b and C3b' receptors of these monocytes showed that the function of both receptors could be dramatically altered by treating the cells with tumor-promoting phorbol esters. Both C3b and C3b' receptors of human monocytes efficiently mediate attachment of erythrocytes coated with the corresponding ligands, but do not promote their ingestion. However, monocytes treated with phorbol myristate acetate (PMA) or phorbol didecanoate ingest C3b- and C3b'-coated erythrocytes. Phorbol esters that are inactive as tumor promoters do not stimulate C3 receptor-mediated phagocytosis. The ability of monocytes to respond to PMA by activation of C3 receptors is developmentally regulated. Freshly isolated monocytes do not take up C3b- or C3b'-coated erythrocytes in response to PMA, but after 3 d of culture they show strong PMA-stimulated uptake. The stimulatory effect of PMA on monocyte C3b and C3b' receptor function occurs within minutes, is stable for hours, is cycloheximide insensitive, and can be inhibited with colchicine. Several lines of evidence indicates that phagocytosis of C3b or C3b'-coated erythrocytes is specifically mediated by the monocytes' C3b and C3b' receptors. First, erythrocytes attached to monocytes with concanavalin A are not ingested when the monocytes are treated with PMA. Second, monocytes plated on IgG-bearing substrates lose Fc receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b- or C3b'-coated erythrocytes after PMA treatment. Third, PMA-treated monocytes plated on C3b-coated surfaces lose C3b receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b'-coated erythrocytes. Conversely, PMA-treated monocytes plated on C3b'-coated surfaces show reduced C3b' receptors activity on their nonadherent surfaces but retain the capacity to ingest C3b-coated erythrocytes.
Asunto(s)
Complemento C3b/inmunología , Monocitos/inmunología , Fagocitosis , Ésteres del Forbol/farmacología , Forboles/farmacología , Receptores de Complemento/efectos de los fármacos , Cationes/farmacología , Células Cultivadas , Colchicina/farmacología , Cicloheximida/farmacología , Eritrocitos/inmunología , Humanos , Fagocitosis/efectos de los fármacos , Podofilotoxina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.
Asunto(s)
Linfocitos B/metabolismo , Complemento C3b/metabolismo , Eritrocitos/metabolismo , Glicoproteínas/farmacología , Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de Complemento/aislamiento & purificación , Animales , Linfocitos B/inmunología , Membrana Celular/análisis , Relación Dosis-Respuesta Inmunológica , Eritrocitos/inmunología , Glicoproteínas/inmunología , Glicoproteínas/aislamiento & purificación , Humanos , Inmunoglobulina G/inmunología , Radioisótopos de Yodo , Neutrófilos/inmunología , Conejos , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/metabolismo , Formación de RosetaRESUMEN
Human leukemic cells were induced to proliferate and mature to macrophage-like cells in primary cultures supplemented with conditioned medium (CM) from phytohemagglutinin and alloantigen-stimulated normal T lymphocytes. Blast and promyelocyte-enriched preparations, isolated after depletion of adherent phagocytic cells and lymphoid cells from samples of myelogenous leukemia patients, were suspended in liquid cultures with 30% CM. Cell cycle analysis was performed throughout the course of induced cellular maturation. Within 24 h of exposure to CM, cells with macrophage-like morphology were identified among the developing adherent cells. Approximately 15-30% of the cells in culture suspensions also developed macrophage-like morphology and esterase reactivity with alpha-napthyl acetate after incubation for 2 d. The number of these nonproliferating cells increased and became predominant in the later culture period. Flow cytometric measurement of DNA content showed that these mature cells had the same aneuploid stemline as the undifferentiated leukemic cells, indicating that genetically abnormal leukemic cells can be induced to differentiate. Reduction in the total RNA content of the macrophage-like cells was also determined by flow cytometry. Reduction in RNA and development of adherent cells served as early markers of maturation, in addition to the later acquisition of complement receptors and phagocytic capacity. Cell cycle analysis showed that CM stimulated the proliferation of immature cells. This initial proliferation may precede intertwined events of proliferation and concurrent maturation of immature cells. Later in the culture period, cellular proliferation decreased, leading to termination of the cultures.
Asunto(s)
Aneuploidia , Transformación Celular Neoplásica/patología , Leucemia Mieloide Aguda/sangre , Adulto , Anciano , Adhesión Celular , Recuento de Células , Ciclo Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Medios de Cultivo/farmacología , ADN/sangre , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Activación de Linfocitos , Macrófagos , Masculino , Monocitos , Fagocitosis/efectos de los fármacos , ARN/sangre , Receptores de Complemento/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal-I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Cuello del Útero/citología , Reposicionamiento de Medicamentos , Células Epiteliales/efectos de los fármacos , Neisseria gonorrhoeae/efectos de los fármacos , Receptores de Complemento/antagonistas & inhibidores , Carbamazepina/farmacología , Células Cultivadas , Farmacorresistencia Bacteriana Múltiple , Células Epiteliales/microbiología , Femenino , Galactosa/metabolismo , Humanos , Metildopa/farmacología , Receptores de Complemento/efectos de los fármacos , Bibliotecas de Moléculas PequeñasRESUMEN
C3bi receptors (CR3) on human polymorphonuclear leukocytes (PMN) bind ligand-coated particles and promote their ingestion. The binding activity of CR3 is not constitutive but is transiently enabled by phorbol esters (Wright, S. D., and B. D. Meyer, 1986, J. Immunol. 136:1759-1764). Our observations indicate that the capacity of CR3 to bind ligand is tightly correlated with the degree of ligand-independent aggregation of the receptor in the plane of the membrane. Fixed PMN were labeled with anti-CR3 monoclonal antibodies and streptavidin colloidal gold before viewing in the electron microscope either en face or in thin section. On unstimulated PMN, gold particles marking CR3 were dispersed randomly. Stimulation of PMN for 25 min with phorbol myristate acetate (PMA) dramatically enhances binding of C3bi-coated particles, and the CR3 on such stimulated cells was observed in clusters containing more than six gold particles. CR3 was not aggregated over coated pits. After 50 min in PMA, the binding activity of CR3 falls, and the distribution of CR3 was again observed to be disperse. If a hydrophilic phorbol ester was washed away after a 20-min stimulation, binding activity remains elevated for at least 50 min, and CR3 remained aggregated. Thus, clustering of CR3 was temporally correlated with its ability to bind ligand and initiate phagocytosis. Unlike CR3, Fc receptors and HLA did not exhibit changes in their aggregation state in response to PMA. Treating PMN with formyl-methionyl-leucyl-phenylalanine, which enhances expression of CR3 but not its function, did not lead to aggregation of CR3. These observations suggest that a clustered configuration is a precondition necessary for binding ligand and signaling phagocytosis.
Asunto(s)
Neutrófilos/inmunología , Receptores de Complemento/metabolismo , Antígenos de Superficie/análisis , Membrana Celular/inmunología , Membrana Celular/ultraestructura , Humanos , Ligandos , Sustancias Macromoleculares , Microscopía Electrónica , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/ultraestructura , Fagocitosis , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento 3b , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Larrea divaricata is a plant widely used in folk medicine in Argentina. This work aimed to study the mechanisms of decoction activity on the release of oxygen reactive species. Decoction increased the binding of zymosan-FITC and superoxide production. Cadmium decreased the superoxide production as well as malonate and barbital. Decoction decreased the release of hydrogen peroxide. Decoction increased the reduction of MTT but not when malonate and barbital were included. Together, decoction increased the expression of dectin-1 leading to increased superoxide production. It is possible that decoction increases the activity of peroxidase, and decreases the Cu, Zn-superoxide dismutase.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Larrea , Macrófagos Peritoneales/efectos de los fármacos , Extractos Vegetales/farmacología , Receptores de Complemento/efectos de los fármacos , Superóxidos/metabolismo , Animales , Barbital/farmacología , Cloruro de Cadmio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Larrea/química , Lectinas Tipo C , Macrófagos Peritoneales/metabolismo , Masculino , Malonatos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Componentes Aéreos de las Plantas , Receptores de Complemento/metabolismo , Zimosan/metabolismoRESUMEN
Human serum lipoproteins are known to participate in or modify several immunologically relevant responses, including the inhibition of target cell lysis initiated by fluid-phase C5b-7 (reactive lysis). We now report that human high density lipoproteins (HDL) can inhibit the complement (C) lytic mechanism after C5b-7, C5b-8, and even C5b-9 have been bound to the target membrane. This inhibitory activity of serum or plasma copurifies in hydrophobic chromatography with antigenically detected apolipoprotein A-I (apoA-I), the major HDL apoprotein, and with HDL in CsCl density gradient ultracentrifugation. Although HDL is more active than its apoproteins in fluid-phase inhibition of C5b-7-initiated reactive lysis, the HDL apoproteins are more effective after C5b-7, C5b-8, or C5b-9 have become bound to human or sheep erythrocytes (E). Highly purified HDL apoproteins, apoA-I and apoA-II, both have greater inhibitory activity than whole HDL on a protein weight basis, and some evidence has been obtained that apoA-I dissociating spontaneously from HDL may be the principal inhibitory moiety in physiological situations. HDL lipids themselves are inactive. The HDL-related inhibitors are ineffective when incubated with EC5b-7 and removed before C8 and C9 are added, and only minimally effective on cell-bound C5b-8 sites before C9 is added. They exert their most prominent inhibitory activity after C9 has been bound to EC5b-8 at low temperature, but before the final temperature-dependent, Zn(++)-inhibitable membrane damage steps have occurred. Therefore, HDL or its apoproteins do not act to repair already established transmembrane channels, but might interfere either with insertion of C9 into the lipid bilayer or with polymerization of C9 at C5b-8 sites. This heat-stable inhibitory activity can be demonstrated to modify lysis of erythrocytes in whole serum, i.e., it does not depend upon artificial interruption of the complement membrane attack sequence at any of the above-mentioned stages. Contributions of the target membrane itself to the mechanism of inhibition are suggested by the observations that, in contrast to sheep or normal human E, lysis of guinea pig E or human E from patients with paroxysmal nocturnal hemoglobinuria is inhibited poorly. This is the first description of a naturally occurring plasma inhibitor acting on the terminal, membrane-associated events in complement lysis. Although further study is required to assess the physiologic or immunopathologic significance of this new function of HDL, the HDL apoproteins or their relevant fragments should be useful experimentally as molecular probes of the lytic mechanism.
Asunto(s)
Apolipoproteínas/sangre , Proteínas Inactivadoras de Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Lipoproteínas HDL/sangre , Animales , Apolipoproteína A-I , Apolipoproteína A-II , Apolipoproteínas/farmacología , Complemento C9/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento/biosíntesis , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Cobayas , Hemólisis , Humanos , Lípidos/sangre , Lipoproteínas HDL/farmacología , Receptores de Complemento/efectos de los fármacos , OvinosRESUMEN
To investigate the greater enzymatic activity of the alternative pathway convertase (and the subsequent greater fixation of C3b) on paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, we have examined the topography of binding of C3b to PNH and normal erythrocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the alpha-chain of C3b was found to bind via predominantly ester bonds to free hydroxyl groups on glycophorin-alpha, the major erythrocyte sialoglycoprotein. The pattern of binding of nascent C3b was the same for normal and PNH erythrocytes. Thus, although C3b binding to a different membrane constituent did not appear to account for the greater enzymatic activity of the alternative pathway convertase when affixed to PNH erythrocytes, it seemed possible that the glycoproteins to which C3b bound might be qualitatively abnormal on the PNH cells, and that structural differences in these molecules might impose modifications in the enzyme-substrate interactions of the alternative pathway convertase. Using methods for radiolabeling both protein and carbohydrate residues, we therefore compared the electrophoretic pattern of the cell-surface glycoproteins on PNH and normal erythrocytes. The glycophorin-alpha dimer was found to be qualitatively abnormal on the PNH cells as evidenced by its greater susceptibility to trypsin-mediated proteolysis. In addition, the abnormal erythrocytes from patients with PNH had fewer periodate oxidizable constituents than did normal erythrocytes, indicating a relative deficiency of cell-surface sialic acid. These investigations suggest that abnormalities in membrane glycoproteins may underlie the aberrant interactions of complement with the hematopoietic elements of PNH.
Asunto(s)
Membrana Eritrocítica/metabolismo , Glicoforinas/metabolismo , Hemoglobinuria Paroxística/sangre , Sialoglicoproteínas/metabolismo , Convertasas de Complemento C3-C5/metabolismo , Vía Alternativa del Complemento , Electroforesis en Gel de Poliacrilamida , Membrana Eritrocítica/análisis , Humanos , Hidroxilamina , Hidroxilaminas/farmacología , Péptidos/sangre , Receptores de Complemento/análisis , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento 3b , Tripsina/farmacologíaRESUMEN
The iC3b receptor (CR3) is required for neutrophil adhesive functions, including homotypic aggregation. Because stimuli that enhance neutrophil adhesion also induce up-regulation of surface CR3, it is widely held that these two responses are causally related. We have dissociated CR3 display (immunofluorescence) from CR3 function (aggregation). Neutrophils isolated at 4 degrees C and rewarmed to 37 degrees C up-regulated surface CR3 twofold, but did not aggregate. The kinetics of FMLP-induced CR3 up-regulation were discordant with those of aggregation. In the absence of extracellular divalent cations, CR3 expression increased twofold after exposure to FMLP, but neutrophils did not aggregate. FMLP elicited 3.5-fold more aggregation than the ionophore A23187, yet less than one-half as much CR3 up-regulation. 3 mM sodium salicylate inhibited aggregation 55 +/- 4%, but had no effect on CR3 up-regulation. Conversely, 1 mM tetracaine completely inhibited CR3 up-regulation, while significantly enhancing aggregation. Neutroplasts expressed CR3, but did not up-regulate the receptor; in contrast, FMLP induced CR3-dependent aggregation of neutroplasts. We conclude that, although constitutive surface CR3 is required for neutrophil aggregation, the up-regulation of CR3 is neither necessary nor sufficient to promote cell-cell adhesion.
Asunto(s)
Agregación Celular , Complemento C3/metabolismo , Neutrófilos/metabolismo , Receptores de Complemento/metabolismo , Anticuerpos Monoclonales/fisiología , Calcimicina/farmacología , Cationes Bivalentes , Agregación Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiología , Citoplasma/metabolismo , Citoplasma/fisiología , Humanos , Cinética , Leucotrieno B4/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/inmunología , Receptores de Complemento 3b , Salicilato de Sodio/farmacología , TemperaturaRESUMEN
Phagocytosis of C3bi- or IgG-opsonized yeast particles in human neutrophils was found to be associated with an increased formation of inositol phosphates and diacylglycerol. Pertussis toxin only marginally affected phagocytosis of IgG- and C3bi-opsonized particles and the associated formation of second messengers. Forskolin, which induced a threefold rise of cellular cAMP, however, markedly inhibited both C3bi- and IgG-mediated phagocytosis as well as the particle-induced formation of inositol phosphates and diacylglycerol. These observations are in contrast to what was found to occur with chemotactic factors and indicate that chemotactic and phagocytic signaling can be regulated independently in human neutrophils. Since C3bi-mediated phagocytosis has been shown to occur at vanishingly low cytosolic free calcium levels, calcium-depleted cells were used to study the importance of the inositol cycle for the engulfment of C3bi-opsonized particles. Despite a total lack of receptor-induced formation of inositol phosphates, a significantly increased accumulation of diacylglycerol accompanied the ingestion of C3bi-opsonized particles. These data show that the engulfment of C3bi-opsonized particles can occur independently of both a calcium transient and an increased inositol phosphate production. However, the observed accumulation of diacylglycerol, not derived from phosphoinositides, suggests that this second messenger play a role in the control of the engulfment process.
Asunto(s)
Antígenos de Diferenciación/fisiología , Calcio/metabolismo , Diglicéridos/biosíntesis , Glicéridos/biosíntesis , Fosfatos de Inositol/biosíntesis , Neutrófilos/fisiología , Fagocitosis , Receptores de Complemento/fisiología , Receptores Fc/fisiología , Fosfatos de Azúcar/biosíntesis , Antígenos de Diferenciación/metabolismo , Colforsina/farmacología , Complemento C3b/metabolismo , Citosol/metabolismo , Diglicéridos/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Fosfatos de Inositol/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Proteínas Opsoninas , Toxina del Pertussis , Fagocitosis/efectos de los fármacos , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento 3b , Receptores Fc/metabolismo , Receptores de IgG , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Z39Ig is a transmembrane protein containing two Ig homology domains with unknown functions. Immunohistochemical analyses of human carotid atherosclerotic plaques detected Z39Ig staining in areas rich in foamy macrophages. Z39Ig staining was also observed in macrophages in the lining layers and sublining areas of rheumatoid arthritis synovium. Z39Ig staining in the osteoarthritis synovium was restricted to macrophages in the lining layers. To identify the role(s) of Z39Ig in the function of macrophages, we used human monocytic cell lines TF-1A (Z39Ig-negative) and THP-1 (Z39Ig-positive). The expression of Z39Ig was induced in TF-1A cells ,when they were differentiated into macrophages by treatment with PMA. The stimulation of PMA-treated TF-1A or THP-1 cells with immobilized anti-Z39Ig mAb induced the secretion of IL-8 and matrix metalloproteinase (MMP)-9, which was dependent on NF-kappaB activation. These data indicate that the macrophage Z39Ig is involved in the pathogenesis of inflammatory diseases through chemokine induction, which will promote the migration of inflammatory cells into the lesion area, and MMP-9 induction, which will contribute to cartilage destruction or extracellular matrix degradation.
Asunto(s)
Artritis/inmunología , Aterosclerosis/inmunología , Macrófagos/inmunología , Receptores de Complemento/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Reacciones Antígeno-Anticuerpo , Línea Celular , Humanos , Inmunohistoquímica , Inflamación , Interleucina-8/metabolismo , Macrófagos/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/inmunología , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
OBJECTIVES: Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. METHODS: We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, ß1, ß2, ß3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. RESULTS: We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances ß-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving ß-adrenergic and C3a receptor activation without associated adverse metabolic effects. CONCLUSIONS: In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules.
Asunto(s)
Adipocitos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Receptores de Complemento/efectos de los fármacos , Células 3T3-L1 , Adipocitos/metabolismo , Adrenérgicos/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular , Lipólisis/efectos de los fármacos , Lipólisis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Obesos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Neuropéptidos/metabolismo , Obesidad/inducido químicamente , Obesidad/metabolismo , Fragmentos de Péptidos/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Complemento/metabolismo , Transducción de Señal , Esterol Esterasa/efectos adversosRESUMEN
The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.
Asunto(s)
Macrófagos/metabolismo , Microglía/metabolismo , Retina/inmunología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Agonistas de Aminoácidos Excitadores/toxicidad , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunohistoquímica , Ácido Kaínico/toxicidad , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Microglía/efectos de los fármacos , Microglía/inmunología , Microscopía Inmunoelectrónica , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Wistar , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/metabolismo , Retina/citología , Retina/lesionesRESUMEN
Cellular damage and inflammatory processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-alpha-lysophosphatidylcholine, a decomposition product of phosphatidylcholine, greatly stimulates mouse peritoneal macrophages to ingest target cells via the Fc receptors. Similarly, treatment of mice with L-alpha-lysophosphatidylethanolamine and L-alpha-lysophosphatidyl-L-serine resulted in an enhanced ingestion activity of macrophages. Cancer cell membranes contain alkyl ether derivatives of phospholipids and neutral lipids. Inflamed cancer cells release decomposition products of alkyl ether phospholipids and neutral lipids, alkyl-lysophospholipids and alkylglycerols, respectively. Administration of alkyl ether analogues of lysophospholipids into mice were able to induce stimulation of macrophages for ingestion with Fc receptor preference. Two synthetic alkylglycerols, dodecylglycerol and tridecylglycerol, were tested. Dodecylglycerol induced an efficient stimulation of macrophages for Fc-mediated ingestion whereas tridecylglycerol induced a minimal level of activation. Therefore, in vivo effect of dodecylglycerol on macrophage stimulation is similar to that of lysophospholipids and their alkyl analogues. These in vivo stimulations of macrophages for Fc receptor-mediated ingestion activity were reproduced in in vitro activation of macrophages by treatment of peritoneal cells with the alkyl lipid derivatives. Among these compounds, dodecylglycerol was found to be the most potent agent for macrophage stimulation. Since macrophages are antigen-presenting cells, the degradation products of cancer cell membrane lipids may have immune potentiating capacity.
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Éteres/farmacología , Lípidos/farmacología , Activación de Macrófagos/efectos de los fármacos , Fosfolípidos/farmacología , Animales , Femenino , Glicerol/farmacología , Técnicas In Vitro , Lisofosfolípidos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento 3b , Receptores Fc/efectos de los fármacosRESUMEN
Fcgamma receptor (FcgammaR)-mediated phagocytosis by mononuclear phagocytes is an essential function in host defense. This process is initiated by crosslinking of membrane FcgammaRs, which induces phosphorylation and activation of Src and Syk tyrosine kinases. Activation of these enzymes is essential for initiating the biochemical cascade that results in the cytoskeletal and membrane changes involved in phagocytosis. Phagocytic capacity and other effector functions of mononuclear phagocytes change during differentiation/maturation of these cells. This is a complex process governed by different soluble and micro-environmental factors, giving rise to populations of cells with distinct phenotypic characteristics. Several agents, including calcitriol, have been shown to induce in vitro differentiation-related phenotypic changes in monocytic cell lines. In this paper, we characterized the changes in the initial biochemical signals associated with the increase in FcgammaR-mediated phagocytosis induced by calcitriol in monocytic U-937 cells. The 10-fold increase in phagocytic capacity is not accompanied by an increase in FcgammaR expression. However, the phosphorylation levels of Lyn and Syk after FcgammaRI or FcgammaRII crosslinking are increased after calcitriol treatment. Our results suggest that signaling induced by FcgammaR in mononuclear phagocytes is not only dependent on the quantity of FcgammaRs aggregated by a stimulus, but it is highly dependent on the cell's differentiation state.
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Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Monocitos/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Precursores Enzimáticos/biosíntesis , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Receptores de Lipopolisacáridos/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Fagocitosis/efectos de los fármacos , Fosforilación , Proteínas Tirosina Quinasas/biosíntesis , ARN/aislamiento & purificación , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/metabolismo , Receptores de IgG/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Quinasa Syk , Factores de Tiempo , Células U937 , Familia-src Quinasas/biosíntesisRESUMEN
In the present study, we investigated the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and which types of neuronal cells contain CaMK II and phosphorylated CaMK II (p-CaMK II) in the CA3 hippocampal region of mice using confocal immunofluorescence study. KA increased the CaMK II, p-CaMK II, glial fibrillary acidic protein (GFAP) and complement receptor type 3 (OX-42) immunoreactivities (IR) at 30 min after KA treatment in mouse hippocampal area. In studies, nevertheless KA-induced CaMK II is expressed in neurons or astrocytes or microglia, p-CaMK II is expressed only in neurons. Thus, our results suggest that the activated CaMK II in early time may be performed important roles only in neurons but not in the astrocytes and microglia.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Ácido Kaínico/farmacología , Neuronas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Hipocampo/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Microscopía Confocal , Neuronas/efectos de los fármacos , Fosforilación , Receptores de Complemento/biosíntesis , Receptores de Complemento/efectos de los fármacosRESUMEN
The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 x 10(-8) M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37 degrees C, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 x 10(-6) M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.